Orange-spotted grouper nervous necrosis virus-encoded protein A induces interferon expression via RIG-I/MDA5-MAVS-TBK1-IRF3 signaling in fish cells

ABSTRACT Nervous necrosis virus (NNV), a highly contagious fish virus, has caused huge economic losses to the global aquaculture industry. A previous study showed that protein A (ProA) encoded by orange-spotted grouper NNV triggers type I interferon (IFN) production in fish cells, but the activation and modulation of correlative signal pathways remain unclear. Here, we proved that ProA induces fish cell-specific IFN promoter activation in a dose-dependent manner. In channel catfish ovary (CCO, an NNV-permissive cell), ProA evoked expression and secretion of functional IFN with anti-DNA virus activity, suggesting a good model for IFN signaling research. A contrastive study of signaling in CCO and fathead minnow (FHM, an NNV-nonpermissive cell) using RNAi knockdown or dominant negative mutant overexpression verified that ProA-mediated IFN activation went through retinoic acid-inducible gene I (RIG-I)-like receptor (RLR) pathway (RIG-I/MDA5-MAVS-TRAF3-TBK1-IRF3) while NOD1-RIPK2-NFκB and TLR3-TRIF branches were unnecessary. As RNA receptors upregulated by ProA in FHM, RIG-I and MDA5 promoted ProA-mediated IFN activation to form a positive feedback loop, while LGP2, NOD1, and PKR inhibited this activation as negative modulators. In ProA-expressed, NNV-infected CCO, the transcription of RIG-I, MDA5, MAVS, TBK1, IRF3, and IFN was downregulated, but the expression of LGP2, TRAF3, and NOD1 remained unchanged, suggesting unknown IFN suppression mechanism by NNV infection. IFN inhibition by overexpressing mutated RIG-I greatly enhanced NNV replication in FHM, implying that RIG-I might be the main target for both ProA-mediated activation and NNV infection-induced inhibition. This study provides overviews and foundations for understanding the interaction between betanodavirus-encoded protein and fish innate immune signaling. IMPORTANCE As a major pathogen, nervous necrosis virus (NNV) infects more than 120 fish species worldwide and is virulent to larvae and juvenile fish, hampering the development of the fish fry industry. Understanding virus-host interaction and underlying mechanisms is an important but largely unknown issue in fish virus studies. Here, using channel catfish ovary and fathead minnow cells as models for the study of innate immunity signaling, we found that NNV-encoded ProA activated interferon signaling via the retinoic acid-inducible gene I (RIG-I)-like receptor (RLR) pathway which was still suppressed by the infection of wild-type NNV. This finding has important implications for the comprehension of NNV protein function and the immune response from different cells. First, RIG-I is the key node for anti-NNV innate immunity. Second, the response intensity of RLR signaling determines the degree of NNV proliferation. This study expands our knowledge regarding the overview of signal pathways affected by NNV-encoded protein and also highlights potential directions for the control of aquatic viruses.


Introduction
Line 86.Please provide the full name of the virus as is the first time is cited in the text.In addition, redaction should include that NNV belongs to the G. Betanodavirus.Line 87 Please delete " a kind of" Line 100 cell lines are susceptible or not to a virus Results Line 26.This sentence is a deduction from the obtained results.It fits better in discussion.Line 155.The authors should provide the TCID50 values obtained after each treatment.Line 181.The authors may choose to state that the titer was calculated by TCID50 method or not to state the method.Line 206 Please delete "medium" Line 233-234 and figure 3H.Although it is true that both mutations have a strong effect on IFN expression I would not say that MDA5 is essential because IFN promoter activity is detected.Please revise this statement Line 269.No DN overexpression is observed in figure 4D, I see a down-regulation when MAVS-DN is used Lines 300.The authors should indicate that up-regulation in CCO is much lower than in FHM.In fact, I'm quite surprised that so low differences in mRNA expression in TBK1 expression in CCO are considered significant.Lines 316-317.The signal pathway is clear in FHM cells, but I do not see so clearly the TBK1 in CCO cells.Discussion As a general comment, I recommend reminding briefly which mutants are being discussed .In addition, I miss more detailed discussion about CCO results Lines 456-458.This sentence is difficult to understand, please re-write .Line 458-460 "The RLR signal pathway...overexpression of the key factors" Please re-redact this sentence.The experimental support of the need of the RLR signal pathway is the knockdown or reduction the gene expression when siRNAs or negative mutants are used.Line 506-510.Please re-write this sentence.It is too long and contains some grammatical errors Line 511-513.To support viral growth a cell must be susceptible (receptors that allow viral attachment are displayed on its surface) and permissive (it allows the completion of the viral cycle and the release of viral progeny) to the virus.Line 516.I do not understand this last assumption.Why do the authors think that NNV RdRp uses as template the host DNA?All single stranded viruses as NNV form double stranded intermediates during its replication process that I guess could be recognized by RIG-like receptors M&M Lines 528-29 full name of SB and ZF4 cells should be provided.

Figure legends
The complete names of the mutants should be provided to make easier interpretation of the results shown in them.Major comments for authors: Huang et al. performed this study to demonstrate orange-spottd grouper nervous necrosis virus-encoded protein A induced IFN via RIG-I/MDA-5 signaling in fish cells.Though the topic is interesting, the reviewer has some questions on statistical analysis and experimental design.Though the PAMPs for proA were determined, the key regulators are still not found.The study is kind of superficial.Some detailed comments can be found below: Fig. 1A, "the universal characteristic IFN activation among fish cells" is an overstatement as some fish cell lines just showed less than 2 fold increase.< 2 fold increase should not be statistically significant but the authors indicated they are (P = 10^-5).Please explain.

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Reviewer(s)' Comments to Author:
Reviewer #1 (Comments to the Author) The manuscript provides interesting information on interferon production against NNV infection in susceptible and non-susceptible cell lines.The study is well designed and appropriate methodology has been used.However, not wanting to make light of the authors' explanation about the need of the RLR signal pathway for IFN stimulation in FHM cells (which do not support NNV replication), I think that more discussion should be focused on the differential response in CCO cells (which does support NNV growth).
Response: Thank you very much for the valuable comments and suggestions.The revised manuscript has been improved substantially with your help.More discussion about IFN response in CCO cells was added to enrich the CCO part.(Line 522-542 in the "Marked-Up

Manuscript")
The source idea of this manuscript was motivated by the results of ProA-induced IFN promoter with huge differences in FHM and CCO cells.To reveal why the IFN response to ProA was so weak in CCO (the cells support NNV growth), we used FHM (the cells do not support NNV growth but exhibit strong IFN response to ProA) as basic cells and performed comparative analysis to derive the immunity of CCO cells based on the results of FHM cells.Some more detailed comments that I think can help to improve the manuscript.

Introduction
(1) Line 86.Please provide the full name of the virus as is the first time is cited in the text.In addition, redaction should include that NNV belongs to the G. Betanodavirus.
Response: We apologize for the unclear abbreviation.In this revised manuscript, the full name and taxonomy of NNV were added and the sentence has been rephrased as "Nervous necrosis virus (NNV) belongs to the family Nodaviridae genus Betanodavirus and is one of the most harmful fish viruses, leading to almost 100% mortalities in juveniles and larvae when infected."(Line87-88 in the "Marked-Up Manuscript") (2) Line 87 Please delete " a kind of" Response: Thanks for the suggestion.The phrase "a kind of" was deleted.
(Line 89 in the "Marked-Up Manuscript") (3) Line 100 cell lines are susceptible or not to a virus Response: Thank you for the suggestion.We are so sorry for the confusion in our previous draft.Actually, NNV can enter both FHM and CCO cells, suggesting that these two cell lines are susceptible to NNV.However, NNV cannot replicate in FHM and only a low level of CP expression can be detected in RIG-I-(Δ1-217) transfected FHM.
Therefore, only CCO but not FHM is permissive to NNV.Following the suggestion, all the "sensitive" have been amended to "permissive" in the abstract (Line 31, 33 in the "Marked-Up Manuscript"), in the text (Line 101,102,205,270,271,467,510 in the "Marked-Up Manuscript"), and in the figure legends (Line 1010 in the "Marked-Up Manuscript").

Results
(4) Line 126.This sentence is a deduction from the obtained results.It fits better in discussion.
Response: Thank you for the suggestion.This sentence was remended to "Furthermore, ProA-triggered IFN promoter activation may be specific for fish cells.34,41;266,272,273,377,378,404,466,498,[577][578][579][580][581][582][583][923][924]937,940,979,988 in the "Marked-Up Manuscript") The primers used for DN construction were also renamed in Table 2. (Please refer to the "Table .docx"file) Some detailed comments can be found below: 1. Fig. 1A, "the universal characteristic IFN activation among fish cells" is an overstatement as some fish cell lines just showed less than 2 fold increase.< 2 fold increase should not be statistically significant but the authors indicated they are (P = 10^-5).Please explain.
Response: Thanks for the comment.In all figures, data were presented as the mean ± standard deviation.Two-tailed, unpaired Student's t-test was used to calculate the statistical differences (P values) between the two groups.Biostatistical analysis revealed a statistically significant difference, although this difference may generally consider not to be biologically significant.However, dose-dependent experiments revealed that ProA induces species-specific IFN promoter activation in a dose-dependent manner, suggesting that although the elevated fold is less than 2, it is biologically significant.An Excel file containing the original data of Fig. 1 was uploaded (Please refer to the "Fig1 Data-upload.xlsx"file).
2. Fig. 1B, did the authors try high dose of ProA?
Response: Thank you for your comment.We did perform the transfection of all four NNV proteins, CP, B1, B2, and ProA, into 293T at various concentrations to test the expression in preparation for interaction and bulk expression purification.However, only B2 can modulate the IFN response in 293T (suppression).We did not observe activation from a high dose of ProA in 293T.Therefore, ProA is proven to not activate IFN response in 293T.Furthermore, we used hMAVS as a positive control and observed significant activation of ISRE upon transfection with the same plasmid.Interestingly, when using the same dose of ProA, it was able to induce the expression of IFN in different fish cells.
How come they showed P=0.0001?Did the authors perform ANOVA before doing two group comparison?
Response: Thank you for your comment and suggestion.The experiment was independently replicated three times, and the resulting P value was calculated by GraphPad using a two-tailed unpaired Student's t-test.The result was found to be 0.0001.We conducted a reanalysis of our data using one-way ANOVA, which revealed significant differences among the groups as shown in the following snapshot.Consequently, the signal transduction of ProA-mediated IFN response does not go through the TLR-TRIF-NF-κB branch.

Fig 8 .
Iine 432.In this figure no DNs results are shown Reviewer #2 (Comments for the Author):

Fig. 1B ,
Fig. 1B, did the authors try high dose of ProA?Fig. 3A, RIG-I just caused 1.5 fold increase in mRNA expression.How come they showed P=0.0001?Did the authors perform ANOVA before doing two group comparison?Fig 5 is not a surprising finding as RIG or MDA activation may trigger TBK1 phosphorylation and IRF3 activation.

Fig. 7 :
Fig.7: ProA can promote TLR3-regulated IFN activation.But why did the authors stated that TLR3 is irrelevant to ProAmediated IFN activation?

( 19 )
Fig 8. Line 432.In this figure no DNs results are shown Response: Thanks for the suggestion.The figure legend of Fig 8E has been corrected as "CP expression representing NNV replication was evaluated by RT-qPCR in FHM cells transfected with RIG-I, MDA5, EV, RIG-I-(Δ1-217) and MDA5-(Δ1-284) at different infection time points as indicated."(Line 987-989 in the "Marked-Up Manuscript").Reviewer #2 (Comments to the Author) Major comments for authors: Huang et al. performed this study to demonstrate orange-spotted grouper nervous necrosis virus-encoded protein A induced IFN via RIG-I/MDA-5signaling in fish cells.Though the topic is interesting, the reviewer has some questions on statistical analysis and experimental design.Though the PAMPs for ProA were determined, the key regulators are still not found.The study is kind of superficial.Response: Thank you very much for your review and comments.The revised manuscript has been improved substantially with your help.In this manuscript, we found that overexpression of ProA can activate IFN response in fish cells.Combining the results of the previous work(Fish   Shellfish Immunol, 2018, 79:234-243), we deduced that ProA generates dsRNA with its RdRp activity, dsRNA as PAMP is recognized by RLR, the RLR signal pathway is activated, and IFN is expressed finally.For the methods of statistical analysis and part of the raw data please see the detailed responses below.

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